The present invention relates to a method of regenerating an enzymatic catalyst by detachment/attachment of an enzyme on a support, said enzyme being used attached to its support in a catalytic reaction.
Nowadays more and more processes use enzymatic catalysis. In the vast majority of cases these processes require fixation of the enzyme on a particulate solid support, either for batch reactions, or for reactions with a fixed catalyst bed. However, enzyme lifetime is of limited duration and regeneration of catalysts containing enzymes is difficult compared to conventional catalysts. Catalyst regeneration consists of detaching the spent enzyme and then attaching an active enzyme on the support. This operation is often complicated, in particular on an industrial scale where large quantities of enzymes are used and their cost is reflected directly in the price of the manufactured products. In fact, until now, regeneration of an enzyme-based catalyst required emptying said catalyst from the reactors, treating the support of said catalyst to remove the spent enzyme, for example by chemical treatment and/or by calcination, then loading the support back in the reactor and finally attaching fresh active enzyme on the latter to restore the active catalyst. Such a method is laborious and expensive as it greatly increases reactor downtime.
The present invention aims to overcome these drawbacks by offering a method that does not require discharging the catalyst for replacing the enzyme, nor emptying the reactor containing it, which reduces reactor downtime. The present invention therefore relates to a method of regenerating an enzymatic catalyst arranged in a reactor comprising a mineral support based on at least one metal oxide and at least one enzyme, characterized in that it comprises at least one step of detachment of the enzymes from the support by solvation by scavenging the catalyst using at least one ionic surfactant until the spent enzymes have been removed, and at least one step of re-attachment of active enzymes by scavenging of said purified support with at least one solution of active enzymes, these two steps being performed in situ within the reactor.
This method not only gives a saving of time relative to the operations of handling and treatment of the support, but also a financial gain resulting from better optimization of the utilization of enzymatic catalysts. It offers the further advantage of being applicable to all types of supported enzymes and all applications using supported enzymes in reactions by enzymatic catalysis. By “enzyme” is meant a molecule allowing lowering of the activation energy of a reaction and acceleration of the chemical reactions taking place in the medium without altering the equilibrium that has been established. They also offer the advantage that they can be used at ambient temperature.
The step of detachment of the enzymes comprises scavenging the catalyst with an aqueous solution of so-called amphiphilic ionic surfactant, this surfactant being selected from the group consisting of salts of alkyl sulphonates, salts of alkyl sulphates, salts of alkyl sulphosuccinates, salts of alkyl phosphate esters, salts of alkylbenzene sulphonates, and quaternary ammonium salts, used alone or mixed. The ammonium salts are generally selected from the salts of formula N R1R2R3R4+ in which R1, R2, R3 and R4 are alkyl, aryl, aralkyl or cycloalkyl groups comprising from 1 to 30 carbon atoms. Among the latter, the tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, tetrapentylammonium, tetrahexylammonium, benzyltrimethylammonium, benzyltriethylammonium and hexamethionium salts are preferred. The phosphonium salts correspond structurally in all respects to the aforementioned ammonium salts.
During the step of detachment of the enzymes, the quantity of enzymes entrained in the effluent leaving the reactor will be measured by measuring, continuously or discontinuously, its absorbance at a wavelength characteristic of the sought enzyme by UV spectrometry. Typically, the wavelengths corresponding to the enzymes vary from 280 nm (nanometers) to 420 nm. In the case when the enzyme used is haemoglobin, the characteristic wavelength is 404 nm. The quantity of enzymes will decrease in the outgoing effluent as the detachment step proceeds. The concentration of spent enzymes measured by absorbance at a wavelength characteristic of the enzyme in UV spectrometry decreases in the outgoing effluent over the entire duration of said scavenging. The end of this step will be reached when the differential measurement of UV absorbance between the outgoing effluent and the stream entering the reactor becomes zero.
Among the ionic surfactants, the alkali metal and alkaline-earth metal salts of alkyl sulphates are preferred. They are selected from the salts of alkyl sulphates in which each alkyl group comprises from 6 to 20 carbon atoms in a linear or branched paraffinic chain, said chain preferably comprising from 10 to 16 carbon atoms. Preferably, the alkyl sulphate salt is a sodium salt of lauryl sulphate, also called sodium dodecyl sulphate (SDS). The ionic surfactant or surfactants are introduced into the reactor mixed with water, with a concentration preferably in the range from 1 to 50 g/L, more preferably from 1 to 20 g/L.
The method of the invention permits detachment of enzymes forming part of the group consisting of the six classes of enzymes, i.e. the hydrolases, the transferases, the oxidoreductases, the isomerases, the lyases or decarboxylases and the lycases. Among these enzymes, the method is particularly suitable for detachment of oxidoreductases, particularly haemoproteins and more particularly haemoglobin.
The supports allowing detachment and attachment of enzymes according to the method of the invention are preferably amorphous or crystalline mineral supports based on metal oxides selected from the group of crystalline, amorphous or composite materials comprising alumina, silica, zirconia, titanium dioxide or any composite material comprising at least one of these materials, with specific surface area in the range from 200 to 1000 m2/g, preferably from silica and/or alumina with specific surface area in the range from 200 to 600 m2/g. According to the method of the invention, re-attachment of the enzymes after the operation of detachment described above is obtained by scavenging the purified support with an enzyme solution until the concentration of enzyme, i.e. its absorbance measured at a wavelength characteristic of the required enzyme by UV spectrometry, increases in the outgoing effluent, and reaches the same absorbance as in the ingoing stream. The re-attachment step is carried out either immediately or later, with the same type of enzyme or a different type of enzyme.
The step of re-attachment of the enzymes is stopped when the differential measurement of the concentration of enzymes, expressed by their absorbance, measured in the ingoing stream and in the effluent leaving the reactor, becomes zero. In fact, the concentration of the solution of enzymes at the outlet is identical to that of the solution going into the reactor, i.e. they have an identical absorbance. If several enzymes of a different nature but mutually compatible were introduced onto the support, said enzymes being introduced together or sequentially, this would be within the scope of the invention.
In a preferred embodiment of the invention, the method includes, between the step of detachment of the spent enzymes and the step of attachment of the active enzymes, a step of washing the support in the reactor with water to remove the spent enzymes and especially the residual surfactant. During this washing step, the absorbance will be measured by UV spectrometry at the wavelength of SDS (260-280 nm) because it is preferable to remove all of the SDS used for detachment, prior to re-attachment of the enzymes. The differential measurement of absorbance between the outgoing effluent and the ingoing stream will remain high since surfactant alone or mixed with spent enzyme will be detected in the wash solution leaving said reactor. Accordingly, the washing step will come to an end when the differential measurement of absorbance between the two streams becomes zero, whether this relates to the UV absorbance of SDS or that of the enzymes that are detached.
According to one embodiment, the end of washing is reached when the absorbance at the wavelength characteristic of the enzyme in the outgoing effluent from the reactor becomes zero. After the re-attachment step, if we wish to use the regenerated enzymatic catalyst in an organic medium, it may be advantageous to circulate, in the reactor, a water/solvent mixture containing a gradually changing concentration from 100 to 0% of water for 0 to 100% of an organic solvent in said mixture, between the start and the end of drying, with drying corresponding to complete removal of water from the support, and optionally withdraw the surplus of unattached enzymes. This solvent will generally correspond to the solvents selected as reaction mixture, for example ketones, esters and ethers.
A second subject of the invention is the application of the method of the invention to all enzymatic catalysts in which the enzymes are attached to the support by low-energy bonds such as van der Waals bonds, electrostatic bonds or hydrogen bonds. A third subject of the invention is the use, in enzymatic catalysis, of the catalysts regenerated according to the method described above.